Chromosome 6q deletion in precursor T-cell lymphoblastic lymphoma and leukemia of childhood and adolescence [Elektronische Ressource] / by Burkhardt, Birgit

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• Gesundheit

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2007
• Nombre de lectures: 11
• Langue: English

Extrait

Chromosome 6q deletion
in precursor T-cell lymphoblastic lymphoma and leukemia
of childhood and adolescence




Inaugural Dissertation
submitted to the
Faculty of Medicine
in partial fulfillment of the requirements
for the PhD-Degree
of the Faculties of Veterinary Medicine and Medicine
of the Justus Liebig University Giessen









by
Burkhardt, Birgit
of
Stuttgart





Giessen 2006
2




From the Center of Pediatrics
Department of Pediatric Hematology and Oncology
Head: Professor Dr. Alfred Reiter
of the Faculty of Medicine of the Justus Liebig University Giessen





















First Supervisor and Committee Member: Prof. Dr. Alfred Reiter
Second Supervisor and Committee Member: Prof. Dr. Martin Stanulla M. Sc.
Committee Members: Prof. Dr. Dr. Hans Michael Piper
Prof. Dr. Rainer Renkawitz



Date of Doctoral Defense: 16.02.2007
3








To Dorothea Neubert and Holger Burkhardt
4

Table of content

1 Summary (English/German) .......................................................................................10

2 Introduction .................................................................................................................12
2.1 T-cell maturation .........................................................................................................12
2.2 Lymphoblastic lymphoma ..........................................................................................16
2.3 Acute lymphoblastic leukemia...................................................................................20
2.4 Deletions of chromosome 6q .....................................................................................28
2.5 Study objectives..........................................................................................................32

3 Material and methods .................................................................................................33
3.1 Materials.......................................................................................................................33
3.1.1 Reagents for Fluorescence in situ hybridization (FISH) ................................................33
3.1.2 Reagents for DNA preparation ......................................................................................34
3.1.3 Reagents for PCR .........................................................................................................34
3.1.4 Reagents for fragment length analysis..........................................................................34
3.1.5 Patients’ samples ..........................................................................................................35
3.2 Molecular genetic methods ........................................................................................36
3.2.1 Fluorescence in situ hybridization (FISH)......................................................................36
3.2.1.1 Selection of bacterial clones, culture of bacteria and DNA preparation ........................37
3.2.1.2 Nick translation..............................................................................................................39
3.2.1.3 Hybridization of probes..................................................................................................41
3.2.1.4 Fluorescence microscopy..............................................................................................43
3.2.2 Loss of Heterozygosity analysis (LOH) .........................................................................44
3.2.2.1 Preparation of DNA in T-LBL samples ..........................................................................45
3.2.2.2 Preparation of DNA in T-ALL samples50
3.2.2.3 UV spectrometry............................................................................................................50
3.2.2.4 Polymerase chain reaction ............................................................................................51
3.2.2.5 Microsatellite markers ...................................................................................................52
3.2.2.6 Primers for polymerase chain reaction53
3.2.2.7 Polymerase chain reaction conditions...........................................................................53
3.2.2.8 Fragment length analysis ..............................................................................................55
3.2.2.9 Data analysis of LOH results.........................................................................................56
5
3.3 Diagnostics and treatment of patients ......................................................................57
3.3.1 Diagnostics and treatment of T-LBL patients ................................................................57
3.3.2 and treatment of T-ALL patients58
3.4 Statistical analysis ......................................................................................................59

4 Results .........................................................................................................................60
4.1 Results of fluorescence in situ hybridization examinations...................................60
4.2 Characterization of the available samples for LOH analysis ..................................62
4.3 Validation of data - quality and results control ........................................................64
4.4 Deletions of chromosome 6q in T-LBL......................................................................65
4.4.1 Patients' characteristics in T-LBL ..................................................................................65
4.4.2 Frequency of LOH in T-LBL ..........................................................................................67
4.4.3 Common deleted region in T-LBL .................................................................................67
4.4.4 Correlation of LOH results with clinical characteristics in T-LBL ...................................69
4.4.5 Prognostic impact of 6q-LOH in T-LBL..........................................................................70
4.4.6 Correlation of LOH results with available cytogenetic data ...........................................71
4.5 Deletion of chromosome 6q in T-ALL........................................................................72
4.5.1 Frequency of LOH in T-ALL ..........................................................................................74
4.5.2 Common deleted region in T-ALL74
4.5.3 Additional markers to delineate the centromeric breakpoint..........................................76
4.5.4 Correlation of LOH results with clinical characteristics in T-ALL ...................................77
4.5.5 Correlation of LOH results and early treatment response .............................................78
4.5.6 Prognostic impact of 6q-LOH results in T-ALL ..............................................................79
4.6 Delineation of a critical region of deletion................................................................80

5 Discussion ...................................................................................................................85
5.1 Methods and samples.................................................................................................87
5.2 Common deleted regions and prognostic impact of 6q deletions .........................87
5.2.1 Common deleted regions and prognostic impact of 6q deletions in T-ALL ...................88
5.2.2 ns in T-LBL90
5.2.3 Comparison of LOH findings in T-ALL and T-LBL.........................................................94
5.2.4 Conclusions of the study ...............................................................................................96



6

6 References ...................................................................................................................97
6.1 Publications .................................................................................................................97
6.2 Internet .......................................................................................................................116

7 List of abbreviations .................................................................................................117

8 Appendix ....................................................................................................................122
8.1 Detailed treatment plan NHL-BFM 95 for lymphoblastic lymphoma ....................122
8.2 Solutions, buffers, and media ..................................................................................123
8.3 DNA preparation from T-LBL samples ....................................................................125
8.4 ration from T-ALL samples135
8.5 Results of LOH analyses in T-LBL patients............................................................141
8.6 yses in T-ALL patients142
8.7 Acknowledgment..................................